Sample Submission GuidelinesAs a leading provider of NGS services, CD Genomics is providing an integrated portfolio of methylation sequencing services. Whole genome bisulfite sequencing (WGBS) is an effective and reliable strategy to identify individually methylated cytosines on a genome-wide scale. With over 10 years of experience and the state-of-the-art next-generation sequencing platforms, we can totally meet your project requirements and budgets in the exploration of methylome.
What Is Whole Genome Bisulfite Sequencing
In mammals, 5-methylcytosine involves the covalent attachment of a methyl group to the 5’ carbon position of cytosine, conferring an additional ability of signaling and regulatory function. As one of the major epigenetic modifications, 5-methylcytosine plays a significant role in many biological processes, including gene silencing, suppression of transposable and repetitive sequences, genomic imprinting, X chromosome inactivation. Detection and quantification of methylation are critical to understanding gene expression and other processes subjected to epigenetic regulation.
Whole genome bisulfite sequencing is the gold-standard approach to acquiring comprehensive base-pair resolution and quantitative information at most genomic methylated cytosines, allowing for unbiased genome-wide DNA methylation profiling. In the whole genome bisulfite sequencing process, the sodium bisulfite treatment of genomic DNA can convert unmethylated cytosines into uracil, while methylated cytosines keep intact. Following this step, PCR amplification, library preparation, and next-generation sequencing are performed. Finally, through comparisons between untreated and sodium bisulfite-treated sequences, it’s viable to determine which nucleotide sites are methylated.
Advantages of Whole Genome Bisulfite Sequencing
- Highly integrated single-base resolution DNA methylation patterning.
- Providing insights into gene cell-fate commitment and reprogramming, as well as gene regulation.
- Identifying novel epigenetic markers and targets for disease.
How Does Whole Genome Bisulfite Sequencing Work?
Our highly experienced expert team and strict quality control following every procedure ensure the comprehensive and accurate results. Before high-throughput sequencing, the DNA sample is processed with cytosine bisulfite conversion followed by tagging at 5’ and 3’ ends, and the introduction of Illumina adapters by PCR amplification.
Whole Genome Bisulfite Sequencing is a method used to study DNA methylation patterns at single-nucleotide resolution. Here's a simplified explanation of how WGBS works:
DNA Extraction: Genomic DNA is extracted from the sample of interest, such as cells or tissues.
Bisulfite Treatment: The extracted DNA is subjected to bisulfite treatment, where sodium bisulfite converts unmethylated cytosine residues (C) to uracil (U), while methylated cytosine residues remain unchanged. This conversion is a critical step as it allows distinguishing between methylated and unmethylated cytosines.
Library Preparation: A sequencing library is then made using the bisulfite-treated DNA. The DNA is broken up into small pieces in this stage, and unique adaptors are attached to the ends of each fragment.
Sequencing: A sequencing machine is used to perform high-throughput sequencing on the created library. When the DNA fragments are sequenced, the unmethylated cytosines show up as Ts due to the bisulfite conversion, while the methylated cytosines are recognized as Cs.
Data Analysis: Bioinformatics tools are used to evaluate the data after sequencing. The software assesses the methylation state of each cytosine in the genome by comparing the sequencing reads to a reference genome.
Methylation Analysis: To find patterns of DNA methylation throughout the genome, the methylation data is then viewed and analyzed. Researchers can examine how DNA methylation is related to gene expression and different biological processes by measuring the amount of methylation at particular genomic locations, such as gene enhancers or promoters.
Please refer to the article "Principles and Workflow of Whole Genome Bisulfite Sequencing" for more detailed information.
Service Specification
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Sample requirements and preparation
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Sequencing
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Data Analysis We provide multiple customized bioinformatics analyses:
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Analysis pipeline
Benefiting from the cutting-edge next-generation sequencing platforms and rich experience, we guarantee you high-quality data and integrated bioinformatics analyses. If you are interested in what CD Genomics can do with the whole genome bisulfite sequencing, please do not hesitate to contact us. We look forward to moving your project forward.
Reference:
Huang, K., & Fan, G. (2010). DNA methylation in cell differentiation and reprogramming: an emerging systematic view. Regenerative medicine, 5(4), 531-544.
1. What species are suited for whole genome bisulfite sequencing?
The species subjected to genome bisulfite sequencing should meet the following three conditions:
a. Eukaryotes.
b. Its reference genome has been assembled to the scaffold level at least.
c. Relatively complete genome annotations.
2. What are the advantages of whole genome bisulfite sequencing?
The technology of whole genome bisulfite sequencing makes genome-wide methylation profiling possible at the single-base level. The major advantage of WGBS is the ability to evaluate the methylation status of nearly every CpG site, involving low-CpG-density regions, including intergenic “gene deserts”, partially methylated domains and distal regulatory elements. In addition, WGBS can reveal absolute DNA methylation level and methylation sequence context.
For studies interested in regions outside of CpG islands, targeted methods such as RRBS and MeDIP are not applicable, and the most proper choice is probably to be WGBS.
3. What are the applications of whole genome bisulfite sequencing?
WGBS Provides insights into cell fate commitment and reprogramming, gene regulation, and the identification of novel epigenetic markers and targets for disease.
WGBS has become the standard approach in some major epigenome consortiums, such as NIH Roadmap (The NIH Roadmap Epigenomics Mapping Consortium), ENCODE (an integrated encyclopedia of DNA elements in the human genome), Blueprint (to decode the epigenetic signature written in blood), and IHEC (International Human Epigenome Consortium).
Divergent DNA methylation patterns associated with gene expression in rice cultivars with contrasting drought and salinity stress response
Journal: Scientific Reports
Impact factor: 4.259
Published: 09 October 2015
Background
DNA methylation plays a central role in the regulation of gene activity and chromatin structure in response to environmental conditions. The understanding of genome-wide DNA methylation provides insights into the regulatory mechanisms of abiotic stress response/adaptation.
Materials: Three rice (Oryza sativa) cultivars, IR64 (drought and salinity sensitive), Pokkali (salinity-tolerant) and Nagina 22 (drought-tolerant).
Results
Researchers investigated the differences between the three cultivars in DNA methylation patterns, DMRs (Differentially methylated Regions), and gene expression, and further inquired into the underlying correlations.
1. Differential methylation is coupled to differential gene expression in different cultivars.
The genes proximal to hypermethylated DMRs showed lower levels of transcript abundance (downregulation) compared to all genes. The genes proximal to hypomethylated DMRs exhibited similar or moderately higher levels of transcript abundance (upregulation) relative to entire gene set. DNA methylation appeared to be correlated with on/off status of gene expression too for DMRs (14.5% for N22/IR64 and 7.2% for PK/IR64).
Relationship between differential methylation and transcript abundance of protein-coding genes.
2. Differential methylation of genes associated with stress response and epigenetic regulation of gene expression.
The differential methylation level and corresponding differential gene expression of rice genes in N22/IR64 and PK/IR64 are shown in Fig. (a) below. The GO analysis of DMR-associated genes in both N22/IR64 and PK/IR64 revealed a significant enrichment of genes that participate in stress response.
Differential methylation, transcript abundance and GO enrichment of proteincoding genes.
3. Methylation status of TEs is correlated with transcription of proximal protein-coding genes.
The enrichment of TEs was substantially higher for DMR-associated DEGs as compared to non-DEGs for both N22/IR64 (P-value 1.49e-05) and PK/IR64 (P-value 5.26e-05) (a). Significantly higher differences in the methylation level of DMRs are associated with TEs as compared to protein-coding genes for PK/IR64 (b).
Correlation between differential expression and frequency/methylation level of TEs.
Discussion
The results suggest the potential role of cultivar-specific DNA methylation patterns as a crucial regulatory mechanism for sensing and responding to the stress conditions via modulation of stress-responsive gene expression. The investigations suggest that variability for drought or salinity tolerance in rice germplasm rely on the extent and patterns of DNA methylation. And the evidences suggest that DNA methylation plays an important part in abiotic stress response by regulating expression of a suit of stress-responsive genes in rice mostly via methylation or demethylation of proximal TEs.
Reference:
Garg R, Chevala V V S N, Shankar R, et al. Divergent DNA methylation patterns associated with gene expression in rice cultivars with contrasting drought and salinity stress response[J]. Scientific reports, 2015, 5.
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